以粒径10μm、孔径300Å球形硅胶为载体，以γ-缩水甘油醚氧丙基三甲氧基硅烷(KH-560)为中间偶联剂，将4-巯基吡啶键合至硅胶上，制备了高效疏水电荷诱导色谱介质。通过单因素实验，探讨了反应温度、反应时间、KH-560的浓度对活化密度的影响，结果表明，在反应温度为75℃，反应时间为10 h条件下，KH-560的利用率大，可获得较宽范围的活化密度，此外还考察了缓冲液pH、反应温度、反应时间、4-巯基吡啶浓度对偶联密度的影响。结果表明，在pH=7.5的缓冲液，反应温度40℃，反应时间6 h下，4-巯基吡啶利用率高，通过控制4-巯基吡啶的浓度，可获得较宽范围的配基密度。最后,于高效液相色谱条件下，通过等度洗脱，考察了流动相pH对牛血清蛋白和溶菌酶保留因子的影响。并通过梯度洗脱，分离了不同等电点的牛血清蛋白和溶菌酶，验证了该介质的高效疏水电荷诱导色谱行为。%High performance hydrophobic charge induction chromatography (HPHCIC) media was synthesized by using 4-mercapto pyridine, bonded to the spherical silica gel with the diameter of 10 microns and the pore size of 300 angstroms, as carrier, and KH-560 as intermediate coupling agent. Firstly, the effects of reaction temperature, reaction time and the concentration of KH-560 on the activation density of the synthetic materials were investigated through the single factor experiments. The results show that, when the reaction temperature is 75℃ and the reaction time is 10 h, the utilization ratio of KH-560 is the largest, meanwhile the activation density of the media can be obtained in a wide range. In addition, the effects of buffer pH, reaction temperature, reaction time and the concentration of 4-mercapto pyridine on the coupling density of the ligand were investigated as well. The results show that, when pH, reaction temperature and reaction time are 7.5, 40℃and 6 h, respectively, a wide range of ligand density of the media can be obtained through controlling the concentration of 4-mercaptopyridine. Finally, under the conditions of high performance liquid chromatography, the effect of the pH of mobile phase on the retention factor of proteins with different isoelectric points, such as bovine serum albumin and lysozyme, was investigated. The above two proteins can be separated by gradient elution. This work demonstrates that the novel synthetic materials can be utilized as the media of high performance hydrophobic charge induction chromatography to separate proteins.
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